New approaches for better protein voltage sensors

更好的蛋白质电压传感器的新方法

基本信息

批准号：
9358357
负责人：
LAWRENCE B COHEN
金额：
$ 69.29万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-09-30 至 2019-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9358357
关键词：
Action Potentials Acute Address Amino Acid Sequence Anatomy Axon Brain Brain region Butterflies C-terminal Cardiac Cells Characteristics Communities Dendrites Dependence Dependency Development Dimerization Embryo Energy Transfer Epilepsy Fluorescence Fluorescent Probes Goals Image Individual Lateral Light Location Measurement Membrane Membrane Potentials Membrane Proteins Modification Molecular Biology Monitor Movement Mutate Mutation Nerve Neurons Noise Nose Olfactory Receptor Cells Optics Phosphoric Monoester Hydrolases Physiological Population Positioning Attribute Presynaptic Terminals Process Proteins Reaction Time Reporter Reporting Screening Result Side Signal Transduction Specificity Speed Synapses Testing Transfection Virion Zebrafish analog base brain cell cell type design dimer imaging potential improved in vivo millisecond neuronal cell body neuronal circuitry novel novel strategies olfactory bulb glomeruli optical spectra presynaptic response sensor tau Proteins tool voltage voltage sensitive dye

项目摘要

This proposal aims to develop better tools for analyzing brain cells and circuits and for large-scale recordings of brain activity. The currently available tools are relatively primitive in terms of sensitivity and speed. One major function of a neuron is to process electrical signals. Thus a tool that is of particular significance is high speed membrane potential imaging. Genetically encoded fluorescent protein voltage indicators (GEVI's) are a obvious strategic approach for “visualizing the brain in action”. Genetically encoded sensors are especially interesting to neuroscientists because, as proteins, they can be expressed in individual cell types in the mammalian brain. Because each brain region has up to 100 different cell types, sensor expression in a specific cell type is essential for imaging the activity of that cell type. Recently, there has been a dramatic improvement in the signal size of GEVIs (i.e. ArcLight, 40%/100 mv) but ArcLight has a relatively slow response time constant (τ=10 msec). There are now several faster GEVIs (τ=0.3 to 2.0 msec) but they have smaller signal sizes (~10%/100mv). One goal of this proposal is developing a GEVI with both large and fast responses to membrane potential changes. Several probe characteristics other than size and speed are also critically important. One is the wavelength range of excitation and emission. At present many sensors and activators are based on GFP and its analogues. Thus, probes with red excitation and emission spectra would allow simultaneous dual function measurements. One aim is to develop useful red GEVIs. Second, many GEVIs have a sigmoidal fluorescence-voltage relationship. The position of this relationship along the voltage axis can be adjusted via mutations in the voltage sensitive domain of the GEVI. Thus GEVIs can be selective reporters of different ranges of the neuron membrane potential and thereby selective for action potential activity versus subthreshold activity. This selectivity depends on both the voltage at half-maximal activation as well as the steepness of the sigmoidal curve. Lastly, it will be important to target the GEVIs to specific regions of the neuron including cell body, dendritic post-synaptic zones, and presynaptic terminals. All of the probes in the proposal are based on the voltage sensitive domain of a membrane protein (a phosphatase from Ciona or zebrafish) with one or two fluorescent proteins inserted into the N- or C-terminal region. The development of improved GEVIs will involve molecular biology for generating novel probes (bigger, faster, red, targeted anatomically and physiologically) followed by testing in cultured HEK293 cells, acutely dissociated neurons, and zebrafish embryos. Probes that function well in these initial screens will then be incorporated into virus particles for in vivo transfection and measurements in the mammalian brain. Improved and selective GEVIs would be useful to the community carrying out optical measurements of brain activity.

该建议旨在开发更好的工具来分析脑细胞和电路以及用于大脑活动的大规模记录。目前可用的工具在敏感性方面相对原始和速度。神经元的主要功能是处理电信号。该工具特别显着性是高速膜电位成像。遗传编码的荧光蛋白电压指标（GEVI）是“在作用中可视化大脑”的明显战略方法。遗传编码传感器对神经科学家特别有趣，因为作为蛋白质，它们可以在个体中表达哺乳动物大脑中的细胞类型。因为每个大脑区域具有多达100种不同的细胞类型，所以传感器特定细胞类型中的表达对于成像该细胞类型的活性至关重要。最近，GEVI的信号大小有了显着改善（即Arclight，40％/100 MV）但是Arclight的响应时间常数相对较慢（τ= 10毫秒）。现在有几个更快的gevis （τ= 0.3至2.0毫秒），但信号尺寸较小（〜10％/100mV）。该建议的目标之一是开发 GEVI对膜电位变化具有大的和快速的响应。大小和速度以外的几个探针特征也非常重要。一个是兴奋和发射的波长范围。目前，许多传感器和激活剂基于GFP及其类似物。那就是红色兴奋和发射光谱的问题将允许简单的双重功能测量。一个目的是开发有用的红色Gevis。其次，许多Gevis具有Sigmoidal 荧光电压关系。该关系沿电压轴的位置可以通过 GEVI的电压敏感域中的突变。 Gevis可以是不同范围的选择性记者神经元膜电位的潜力，从而选择性活性与亚阈值活性。这种选择性取决于半最大激活时的电压以及Sigmoidal的钢曲线。最后，将GEVIS靶向于神经元的特定区域至关重要，包括细胞体，树突状后突触区和突触前末端。该提案中的所有问题均基于膜蛋白的电压敏感结构域（A 来自ciona或斑马鱼的磷酸酶，插入N-或C末端的一个或两个荧光蛋白地区。改进的GEVI的发展将涉及分子生物学来产生新型探针（更大，更大，更快，红色，在解剖和物理上针对的），然后在培养的HEK293细胞中测试，急性解离神经元和斑马鱼胚胎。然后，在这些初始屏幕中运作良好的探针将是掺入哺乳动物大脑的体内转化和测量中的病毒颗粒中。改进选择性GEVI对进行大脑活动的光学测量将很有用。